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Toxin-antitoxin loci regulate adaptive responses to stresses associated with the host environment and drug exposure. Phylogenomic studies have shown that Mycobacterium tuberculosis encodes a naturally expanded type II toxin-antitoxin system, including ParDE/RelBE superfamily members. Type II toxins are presumably regulated exclusively through protein-protein interactions with type II antitoxins. However, experimental observations in M. tuberculosis indicated that additional control mechanisms regulate RelBE2 type II loci under host-associated stress conditions. Herein, we describe for the first time a novel antisense RNA, termed asRelE2, that co-regulates RelE2 production via targeted processing by the Mtb RNase III, Rnc. We find that convergent expression of this coding-antisense hybrid TA locus, relBE2-asrelE2, is controlled in a cAMP-dependent manner by the essential cAMP receptor protein transcription factor, Crp, in response to the host-associated stresses of low pH and nutrient limitation. Ex vivo survival studies with relE2 and asrelE2 knockout strains showed that RelE2 contributes to Mtb survival in activated macrophages and low pH to nutrient limitation. To our knowledge, this is the first report of a novel tripartite type IIb TA loci and antisense post-transcriptional regulation of a type II TA loci., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low microM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Genetic and biochemical evidence has implicated two different target enzymes for isoniazid (INH) within the unique type II fatty acid synthase (FAS) system involved in the production of mycolic acids. These two components are an enoyl acyl carrier protein (ACP) reductase, InhA, and a beta-ketoacyl-ACP synthase, KasA. We compared the consequences of INH treatment of Mycobacterium tuberculosis (MTB) with two inhibitors having well-defined targets: triclosan (TRC), which inhibits InhA; and thiolactomycin (TLM), which inhibits KasA. INH and TLM, but not TRC, upregulate the expression of an operon containing five FAS II components, including kasA and acpM. Although all three compounds inhibit mycolic acid synthesis, treatment with INH and TLM, but not with TRC, results in the accumulation of ACP-bound lipid precursors to mycolic acids that were 26 carbons long and fully saturated. TLM-resistant mutants of MTB were more cross-resistant to INH than TRC-resistant mutants. Overexpression of KasA conferred more resistance to TLM and INH than to TRC. Overexpression of InhA conferred more resistance to TRC than to INH and TLM. Co-overexpression of both InhA and KasA resulted in strongly enhanced levels of INH resistance, in addition to cross-resistance to both TLM and TRC. These results suggest that these components of the FAS II complex are not independently regulated and that alterations in the expression level of InhA affect expression levels of KasA. Nonetheless, INH appeared to resemble TLM more closely in overall mode of action, and KasA levels appeared to be tightly correlated with INH sensitivity.